Friedreich?s Ataxia (FRDA) is a neurodegenerative disease caused by decreased expression of a mitochondrial protein, frataxin, leading to increased mitochondrial iron, oxidative stress, and loss of iron sulfur cluster synthesis. The YFH1 gene is the Saccharomyces cerevisiae homologue of the human frataxin gene. In collaboration with Dr. Copeland and Dr. Resnick?s laboratories, we have found cells lacking YFH1 exhibit 1) accumulation of iron, which cannot be exported from the mitochondria; 2) oxidation of proteins; 3) oxidative DNA damage, which leads to petite colony formation with defects or loss of mitochondrial DNA and 4) nuclear chromosomal damage. The cellular impact of mitochondrial iron overload in yeast is being determined by global gene expression profiling (in collaboration with the NIEHS Microarray Center) in an yfh1 deletion mutant with defective mitochondrial function and no mitochondrial DNA (i.e., rho0). In order to better replicate the human disease process , we in collaboration with Dr. Mike Resnick also conducted transcription profiling on a yeast strain with a rheostatable system that can lead to a lowering of the expressing of the YFH1 gene. We examined the gene profiles in yeast in which frataxin was reduced twofold to sevenfold in generations 3 through 24, resulted in nearly identical events as those occurring in the knockout experiment. These transpired even with the initial reduction of frataxin at generation 3. Overall, we found the mostly downregulated cytochrome, heme, and iron/sulfur cluster assembly pathways, to indicate that frataxin has a role in iron transport, iron/sulfur cluster biosynthesis, oxidative phosphorylation, and as an antioxidant. Furthermore, we find these data correlate very well with a recently published iron deficiency profile. [unreadable] [unreadable] This inter-institute collaborative project between a clinical laboratory (Drs. Nicholas DiProspero and Kennetth Fischbeck) at NINDS and the Van Houten laboratory at NIEHS seeks to test the hypothesis that FRDA patients will: 1) accumulate mitochondrial DNA damage in peripheral lymphocytes; 2) share common gene expression patterns unique to the pathogenesis of the disease; and 3) show diminution of both of these effects by idebenone treatment. This collaborative project represents new research and would not be possible without a direct collaboration between these two laboratories and is supported by a grant from the Office of Rare Diseases. [unreadable] This study has five specific aims:[unreadable] 1.1. Mitochondrial DNA (mtDNA) damage, an indicator of oxidative stress, will be measured using QPCR in lymphoblastoid cells from patients with FRDA. MtDNA damage following treatment with hydrogen peroxide will be studied. This SA will test the hypothesis that idebenone will lead to a decrease in basal mtDNA damage in FRDA patients and help prevent oxidant injury. [unreadable] 1.2. Gene expression profiling using a 22,000 gene oligonucleotide Agilent chip will be performed on lymphoblastoid cells from FRDA patients and controls will be used to assess global transcriptional changes associated with FRDA. It is hypothesized that a common set of genes will be altered in FRDA patients, and that idebenone treatment will lead to abrogation of these changes as compared to control cell lines. [unreadable] 1.3. Lymphocytes from 48 FRDA patients undergoing a double-blind placebo controlled phase II study will be assessed for mtDNA damage using the QPCR assay. Levels of damage will be measured prior to and after treatment. [unreadable] 1.4. Gene expression profiles of lymphocytes from 48 FRDA patients will be compared to a pool of normal individuals. Blood will be sampled prior to the initiation and after idebenone treatment. [unreadable] 1.5. In collaboration with Icoria global biochemical profiles will be determined, in triplicate, using Icoria?s LC/MS and GC/MS platforms, on patient serum (before and after idebenone treatment). This analysis can measure about ~1,000 components. We hypothesize that most severely affected patients will show higher glycolytic profiles and that idebenone will help reverse this effect. [unreadable] [unreadable] Together these five aims will develop clinically relevant markers for disease progression, identify specific targets for treatment, and assess the effectiveness of idebenone in reversing these markers of disease. The initial analysis of four human lymphoblastoid lines by gene expression profiling has already indicated important global changes in the up regulation of genes encoding glycolytic enzymes and a down regulation of genes encoding TCA cycle enzymes (aconitase and succinate dehydrogenase, which contain FeS centers). Finally, completion of this study will allow us to compare gene expression data from four organisms suffering a pathological decrease in the level of frataxin expression: yeast, C. elegans, Drosophila and humans. These cross species comparisons of data sets will help us understand the common pathways affected by a decrease in frataxin, as well as, give us better insight into this devastating human disease.